Advances in the Enzymatic Synthesis of Nucleoside-5′-Triphosphates and Their Analogs

Abstract

Nucleoside-5′-triphosphates (5′-NTPs) are essential building blocks of nucleic acids in nature and play an important role in molecular biology, diagnostics, and mRNA therapeutic synthesis. Chemical synthesis has long been the standard method for producing modified 5′-NTPs. However, chemical routes face limitations, including low regio- and stereoselectivity, along with the need for protection/deprotection cycles, resulting in low yields, high costs, and lengthy processes. In contrast, enzymatic synthesis methods offer significant advantages, such as improved regio- and stereoselectivity and the use of mild reaction conditions, which often leads to higher product yields in “one-pot” reactions. Despite the extensive review of chemical synthesis routes for 5′-NTPs, there has not yet been any comprehensive analysis of enzymatic approaches. Initially, this review provides a brief overview of the enzymes involved in nucleotide metabolism, introducing valuable biocatalysts for 5’-NTP synthesis. Furthermore, the available enzymatic methods for efficient 5′-NTP synthesis using purified enzymes and starting from either nucleobases or nucleosides are examined, highlighting their respective advantages and disadvantages. Special attention is also given to the importance of ATP regeneration systems for 5′-NTP synthesis. We aim to demonstrate the remarkable potential of enzymatic in vitro cascade reactions, promoting their broader application in both basic research and industry.

1. Background

1.1. Nucleotides in Metabolism and Industrial Applications

Living cells contain a wide variety of nucleotides, which play important roles in various biochemical processes. These molecules participate in key functions such as energy storage, serving as activated precursors for DNA and RNA synthesis (the storage and translation of genetic information), functioning as intra- and extracellular signaling molecules, and acting as antibiotics, coenzymes, or activators and carriers for other compounds like sugars [1,2]. Among the most prevalent nucleotides are 5′-phosphorylated nucleosides, which may have one (nucleoside-5′-monophosphates, 5′-NMPs), two (nucleoside-5′-diphosphates, 5′-NDPs), three (5′-NTPs), or even more phosphate groups attached to the 5′-carbon of the sugar moiety of a nucleoside.

5′-NTPs and 2′-deoxy-5′-NTPs (5′-dNTPs) are the fundamental components of nucleic acids (RNA/DNA), with RNA building blocks typically being 10–100 times more abundant than their deoxynucleotide equivalents in living cells [3]. Among these, adenosine-5′-triphosphate (ATP) is the most abundant nucleotide, with intracellular concentrations ranging from 1–5 mM. ATP serves not only as a building block for RNA but also as a phosphate and energy donor in numerous enzymatic reactions, and acts as a key player in both intra- and extracellular signaling [4,5]. Guanosine-5′-triphosphate (GTP) is critical for cellular signal transduction, particularly in relation to GTP-binding proteins (G-proteins) and G-protein coupled receptors [6]. Additionally, in bacteria, GTP and guanosine-5′-diphosphate (GDP) are involved in the stress adaptation process (stringent response) by serving as precursors for the synthesis of alarmones such as (p)ppGpp [7]. Cytidine-5′-triphosphate (CTP) is essential for lipid synthesis, while a number of 5′-NTPs can combine with sugars to form sugarnucleotides [8]. Sugar nucleotides play a crucial role as intermediates in carbohydrate metabolism and the synthesis of glycoconjugates. These molecules consist of a sugar or sugar derivative linked to a 5′-NDP or 5′-NMP. Examples include UDP-α-D-galactose, GDP-α-D-mannose, or CMP-sialic acid [9].

The production of nucleotides and their analogs has been a major focus of research for decades due to their wide range of applications in pharmaceuticals, molecular biology, and structural chemistry [2,10,11,12,13]. 5′-NTPs are mainly the active forms of most antiviral and anticancer nucleoside drugs. Consequently, nucleotide analogs, which include modifications to the sugar, base, or phosphate components, are used to explore and manipulate biological systems during drug development [14,15]. In addition to nucleoside analogs, protected 5′-NDP-/5′-NTP- derivatives are being studied as potential activated drugs for direct application [16,17]. In molecular biology, 5′-dNTPs and 5´-NTPs are employed as building blocks for DNA and RNA, respectively, in various applications such as PCR, mutagenesis experiments, diagnostic probes, and the study of macromolecular structures and their interactions [12]. Naturally occurring 5′-NTPs like ATP are also used as cofactors in numerous in vitro assays, including phosphorylations, ligations, and cell-free protein expression [5]. ATP is also one of the most important phosphate donors in enzymatic cascade reactions [18]. Moreover, 5′-NTPs are key components for the synthesis of mRNA therapeutics by in vitro transcription, which show great promise for treating a wide range of diseases and serving as vaccines [19]. The development of vaccines for COVID-19 represents one of the most efficient vaccine campaigns in history, with modified 5′-NTPs playing a crucial role by reducing potential toxicity and immunogenicity. Notably, the N1-methylpseudouridine modification significantly improved the efficacy of mRNA vaccines [13]. This makes 5′-triphosphorylated N1-methylpseudouridine and pseudouridine promising nucleotide analogs for future RNA-based therapies and vaccine developments.

1.2. Enzymes Involved in Nucleotide Metabolism

In nature, nucleotides are synthesized through the de novo or the salvage pathway. The de novo synthesis of pyrimidine nucleotides begins with glutamine or aspartate and progresses through orotate and 5-phospho-D-ribosyl-α-1-pyrophosphate (PRPP), ultimately forming orotidine-5′-monophosphate. It is then decarboxylated to form uridine-5′-monophosphate (UMP), which is further converted into UDP, UTP, and other pyrimidine nucleotides, including those with cytosine or thymine bases [3].

In contrast to pyrimidine nucleoside synthesis, where the nucleobase is synthesized separately and then attached to an activated sugar, de novo purine nucleoside synthesis occurs directly on the ribose backbone. Starting with PRPP, an amide nitrogen is first added to the 1-position, followed by the stepwise construction of the purine nucleobase with the help of molecules such as glycine, glutamine, and aspartate [1]. The resulting inosine-5′-monophosphate (IMP) serves as a precursor for other purine nucleotides, including adenosine-5′-monophosphate (AMP), xanthosine-5′-monophosphate, and guanosine-5′-monophosphate (GMP), which are then further phosphorylated to form the respective 5′-NDPs and 5′-NTPs. Deoxyribonucleotides are generated by ribonucleotide reductase (RNR) from their ribose counterparts, primarily at the 5′-NDP level (Figure 1). Detailed reviews have summarized the enzymes involved in the de novo pathway [3].

Certain cell types and even entire organisms, such as many parasites, are incapable of performing the de novo synthesis, particularly of purine nucleotides [20]. Instead, they rely on the salvage pathway, where nucleobases and nucleosides from external sources or internal degradation are recycled to produce nucleotides, requiring less energy input. For nucleotide and nucleoside breakdown, various nucleosidases and phosphatases exist. An interesting example is the nucleosidase PpnN of Escherichia coli, which regulates purine homeostasis by cleaving 5′-NMPs [21]. Its activity is notably stimulated by the alarmones (p)ppGpp. Nucleosidases, nucleotidases, and phosphatases are only included in this review in case they have also been shown to be active in the synthesis direction.

Figure 1 provides an overview of the key enzymes responsible for the formation and interconversion of 5′-NMPs (Section 1.2.1), 5′-NDPs (Section 1.2.2 and Section 1.2.4), and 5′-NTPs (Section 1.2.3 and Section 1.2.4). These enzymes have also been applied in in vitro synthesis routes to produce valuable 5′-NTPs. The focus is on the substrate scope of the enzymes, with information on other aspects such as structure and kinetics being available elsehere [22,23].

1.2.1. Enzymes Involved in the Synthesis of Natural and Modified 5′-NMPs

5′-NMPs can be synthesized from nucleobases or nucleosides, or they can be derived from the enzymatic degradation of RNA and DNA, such as by nucleases [24] (Figure 2). Purine and pyrimidine phosphoribosyl transferases (PRTs), like adenine PRT, hypoxanthine-guanine PRT, uracil PRT, and orotate PRT, catalyze the coupling of a nucleobase with PRPP to form the respective 5′-NMP (Figure 1). The classification of PRTs and their use in the synthesis of various natural and base-modified NMPs was reviewed by Arco and Fernandez-Lucas in 2017 [25].

Nucleosides featuring a C-glycosidic bond instead of the typical N-glycosidic bond, such as pseudouridine-5′-monophosphate, are formed by pseudouridylate synthase (PUS or YeiN). This enzyme couples the nucleobase directly to ribose-5-phosphate (PR) [26] (Figure 1). The authors demonstrated that this enzyme can be applied on a milligram scale to produce a variety of sugar- and base-modified pseudouridine-5′-monophosphate analogs.

Alternatively, 5′-NMPs are generated by the 5′-monophosphorylation of nucleosides through various enzymes and phosphate donors. Nucleoside kinases (NKs) and deoxynucleoside kinases (dNKs) are the most well-known enzymes that catalyze the nearly irreversible 5′-monophosphorylation of (deoxy)nucleosides using 5´-(d)NTPs as phosphate donors (Figure 1). General dNKs accept a broad range of nucleoside substrates, while variants such as adenosine kinase or uridine-cytidine kinase have a narrower substrate spectrum. Due to their crucial role in activating antiviral and anticancer nucleoside drugs in the human body, both human and viral (d)NKs have been extensively studied [22].

Given that most antiviral and anticancer nucleoside drugs are deoxynucleoside analogs, much of the research has focused on dNKs rather than NKs. The broad substrate specificity of human (d)NKs is evident in the variety of nucleoside analog drugs that are activated to 5′-NTPs. These analogs include open-ring sugars like ganciclovir, open-ring bases such as ribavirin and mizoribine, and nucleosides with fluorine, azide, or no functional groups at the 2′ and 3′-positions, such as gemcitabine, zidovudine, and stavudine, as well as those with unusual sugar conformations like cytarabine, arabinosyl guanine, and lamivudine. Other examples include large base modifications like brivudine; halogenated derivatives like cladribine, fludarabine, or 5-fluorouridine; thiolated bases like 2-thiocytidine and 4-thiouridine; and carbocyclic derivatives such as aristeromycin, cyclopentenyl cytosine, or carbocyclic deoxyguanosine [22,27,28,29].

However, not only mammalian cells but also other organisms harbor wide-spectrum enzymes. Methanocaldococcus jannaschii and Thermoplasma acidophilum possess wide-spectrum NKs that exhibit activity toward inosine, cytidine, guanosine, and adenosine [30,31]. Several insects, including the fruit fly Drosophila melanogaster (DmdNK), silk moths, and mosquitoes, have multisubstrate dNKs [32]. DmdNK, with its remarkable substrate spectrum and activity, has been the focus of extensive research [23,33,34,35] and has been applied in the synthesis of various NMP analogs [36]. Additionally, viral dNKs, such as the thymidine kinase of Herpes simplex virus (HSV), exhibit broad substrate specificity, making them another important area of study.

In addition to (d)NKs, several other enzymes are involved in the phosphorylation of nucleosides and their analogs, including 5′-nucleotidases [37,38], nonspecific acid phosphatases [39,40] or nucleoside phosphotransferases [41], and even protein kinases. It is worth highlighting nonspecific acid phosphatases here. Two types of bacterial nonspecific acid phosphatases, also known as acid phosphatase/phosphotransferase, have the ability not only to dephosphorylate but also to synthesize 5´-NMPs at optimal pH values of 5 or below [39,42]. Unlike transferases, these hydrolases do not require organic cofactors as phosphate donors. For example, Yamada’s group demonstrated the conversion of natural ribonucleosides into their corresponding 5′-NMPs using inorganic pyrophosphate as the phosphate donor [43]. However, due to the concurrent phosphatase activity of nonspecific acid phosphatases, NMP yields were moderate, reaching a maximum of 41% with the Morganella morganii enzyme [43] and 45.5% with the Escherichia blattae enzyme [44]. Through random mutagenesis of the Morganella morganii enzyme, IMP production from inosine and inorganic pyrophosphate was increased from 49% to 88% due to its reduced phosphatase activity [45]. Later, using whole Escherichia coli cells expressing the modified gene, more than 99% IMP (around 120 mM and 47 g/L) was synthesized [46].

Additionally, 5′-NMPs can be interconverted through the modification of functional groups by various enzymes, such as AMP deaminase (AMP to IMP), IMP dehydrogenase (IMP to xanthine-5′-monophosphate), and GMP synthase (xanthine-5′-monophosphate to GMP) (Figure 1). Interconversions also occur at the nucleobase or nucleoside level (Figure 1). Adenosine deaminase is commonly employed in nucleoside synthesis, enabling the production of soluble adenosine derivatives via transglycosylation, which can then be converted into less soluble inosine or guanosine compounds [47]. Another interesting interconversion at the nucleoside level is catalyzed by the pyrimidine nucleoside 2′-hydroxylase (PDN2′H) from Neurospora crassa [48]. It naturally hydroxylates thymidine at the α-2′-position to give 5-methyluridine as a product. The enzyme is an α-ketoglutarate/Fe(II)-dependent dioxygenase.

1.2.2. Enzymes Synthesizing 5′-NDPs and Their Analogs

Most 5′-(d)NDPs are synthesized from 5′-(d)NMPs by NMP kinases (NMPKs) in a reversible reaction, using 5′-(d)NTP as the phosphate donor (Figure 1). NMPKs are typically specialized for substrates or substrate groups, such as adenylate kinase or UMP-CMP kinase [49]. Unlike (d)NKs, NMPKs mainly do not differentiate between ribo- and deoxyribo-substrates [50]. However, a small group of dNMPKs, such as those found in bacteriophages T4 and T5, are specific to deoxynucleotide substrates while accepting a broad range of nucleobases [51]. Viral and parasitic NMPKs often exhibit a broad substrate range. For example, thymidylate kinase from Plasmodium falciparum phosphorylates dGMP, dUMP, dIMP, and GMP, in addition to thymidine-5′-monophosphate (TMP). Similarly, the thymidylate kinase from Vaccinia virus has a wide substrate spectrum, phosphorylating TMP, UMP, halogenated UMP derivatives, and bulkier substrates like dGMP or brivudine monophosphate [22,52].

The interconversion of 5′-NDPs to 5′-dNDPs, specifically at the 2′-hydroxyl group, is catalyzed by RNRs (Figure 1). RNRs play a crucial role in DNA synthesis across all domains of life and are present in many viruses as well [53].

1.2.3. Enzymatic 5´-NTP Synthesis

The primary route for 5′-(d)NTP synthesis involves the phosphorylation of 5′-(d)NDPs by NDP kinases (NDPKs), which utilize other 5′-(d)NTPs as phosphate donors (Figure 1). NDPKs demonstrate low specificity for both the phosphate acceptor and donor. A wide range of nucleobases are accepted, as the binding residues form a cleft with aromatic stacking, rather than making specific polar interactions with surrounding amino acids [54,55]. There are minimal restrictions on the 2′-position of the sugar moiety, allowing both ribose and deoxyribose nucleotides to be converted. However, the presence of the 3′-hydroxyl group is essential for efficient performance [54]. Derivatives with modifications on the phosphate groups, such as thio-phosphates [56] or sulfuryl-groups [57], have been shown to be weak substrates. A wide variety of modified 5′-NTPs have been produced using NDPKs so far, including 8-hydroxy-dGTP, 2-hydroxy-dATP [58], five azole carboxamide 5′-dNTPs [59], and pyrimidine and purine 5′-NTPs such as arabinosyl CTP, 2′-deoxy-2′-azido-CTP and -UTP, 2′,3′-dideoxy-3′-azido-dATP, and 2′-deoxy-2′-amino-GTP [60].

Other widely used enzymes for 5′-NTP synthesis include pyruvate kinase (PK), acetate kinase (AcK), and creatine kinase (CK), which are also commonly used in in vitro (d)ATP regeneration systems. These enzymes show a broad substrate spectrum and have been employed to synthesize a variety of 5′-NTPs and their analogs. PK, using phosphoenolpyruvate as the phosphate donor, was able to generate all natural 5′-(d)NTPs from the respective 5′-(d)NDPs [61]. Additionally, it was used for synthesizing ribavirin-5′-TP [62], arabinosyl CTP [63], and 8-azaguanosine-5′-triphosphate [64]. CK, using phosphocreatine as phosphate donor, was applied for the synthesis of ß-L-2′,3′-dideoxy-ATP [65] and 2-fluoro-ATP [66].

When Escherichia coli cells were found to remain viable even with disrupted NDPK and PK genes, it was also discovered that NMPK exhibited NDPK activity [67]. Further research demonstrated NDPK activity in human UMP-CMP kinase and adenylate kinase 9, which are capable of converting 5′-(d)NMPs sequentially to the corresponding 5′-(d)NTPs [68]. However, this capability has not yet been utilized in 5′-NTP production processes.

Interconversions between 5′NTPs are also possible, as seen in the UTP to CTP conversion catalyzed by CTP synthetase (Figure 1). Additionally, some nucleoside-specific deaminases have demonstrated minor activity toward 5′-NTPs [69].

1.2.4. Conversion of 5′-NMPs and 5′-NDPs to 5′-NTPs by Polyphosphate Kinases

Polyphosphate kinases (PPKs) catalyze the conversion of 5′-NMPs and 5′-NDPs into 5′-NTPs, utilizing polyphosphate as a cost-effective source of inorganic phosphate (Figure 1). This ability makes PPKs valuable enzymes for ATP regeneration starting from AMP or ADP. Recent reviews have highlighted the use of PPK enzymes for 5′-NTP and 5′-NDP regeneration in coupled enzymatic reactions [70,71].

In 2014, a classification of PPKs was proposed based on phylogenetic and functional analyses, categorizing these enzymes into two distinct families with no sequence similarity [72]. PPK1 enzymes generally favor polyphosphate synthesis, while PPK2 enzymes are inclined toward polyphosphate degradation. The PPK2 family was further divided into three subclasses: class I, which involves 5′-NDP phosphorylation; class II, which is associated with 5′-NMP phosphorylation (also known as PAP (polyphosphate:AMP phosphotransferase)); and class III, where both 5′-NMP and 5′-NDP phosphorylation is observed. However, due to numerous exceptions, predicting an enzyme’s substrate specificity based solely on its sequence has proven unreliable [5]. As a result, Andexer’s group suggested that this classification should be viewed primarily as reflecting the enzymes’ preferences, as many enzymes have been shown to catalyze all types of reactions [73].

Escherichia coli PPK is one of the most well-studied enzymes in the PPK1 family, first identified in the 1950s for its ability to regenerate ATP from ADP in a coupled hexokinase reaction [74]. Notably, research has shown that the 5′-NDP phosphorylating activity of Escherichia coli PPK1 is reversible only with ADP, while phosphate transfer from polyphosphate to other 5′-NDPs occurs irreversibly [75,76]. Initially, PPK1 enzymes were primarily used in ATP regeneration for coupled enzymatic reactions. However, recent focus has shifted toward PPK2 enzymes, especially the class III subtype [70]. Recent studies on novel PPK2 class III enzymes revealed promising candidates, which were successfully applied in a large-scale, coupled carboxylate reduction to aldehyde [71].

PPKs are also known for their broad substrate specificity. For instance, the PPK2 enzyme from Cupriavidus necator (formerly Ralstonia eutropha) exhibits one of the widest substrate spectra recorded, accepting ADP, GDP, CDP, UDP, and TDP as substrates [77]. Escherichia coli PPK1 was shown to phosphorylate all natural NDPs and dNDPs, namely ADP, dADP, dGDP, GDP, TDP, UDP, CDP, and dCDP [76]. The PPK2 class III enzyme from Meiothermus ruber was found to phosphorylate ADP, GDP, CDP, and UDP, in addition to their corresponding NMPs [72]. In general, purine nucleotides were preferred over pyrimidines (AMP > GMP > CMP > UMP > TMP) [78]. Due to their broad substrate range, PPKs have been utilized in enzymatic cascade reactions for the synthesis of both natural and modified 5′-NTPs [66,79,80].

1.3. Chemical Synthesis of NTPs and Their Analogs

Since the first laboratory synthesis of ATP in 1949, researchers worldwide have worked to develop efficient routes for synthesizing 5′-NTPs and their analogs [50]. To this day, the most widely used chemical methods are based on protocols established by Yoshikawa and Ludwig. The Yoshikawa method [81] involves the selective 5′-monophosphorylation of a nucleoside precursor (2,3′-O-iso-propylidene nucleoside) using electrophilic phosphorous oxychloride (POCl₃) [10]. The resulting phosphorodichlorate intermediate is then reacted with pyrophosphate to form the cyclic triphosphate, which is ultimately hydrolyzed to yield the desired product. In contrast, the Ludwig–Eckstein method [82], a “one-pot, three-step” process, employs 3′-O-protected nucleoside precursors (with 2′-O protection for NTPs) [10]. These precursors react with salicyl phosphorochlorite to form an activated phosphite intermediate. Two nucleophilic substitution reactions, initiated by tris(tetra-n-butylammonium) hydrogen pyrophosphate, produce the cyclic intermediate. Finally, iodine-mediated oxidation of this intermediate yields the modified (d)NTP. The Ludwig–Eckstein method offers the advantage of reducing undesired byproducts, simplifying the subsequent HPLC purification process.

The Ludwig method has undergone significant refinement, with considerable efforts directed at improving its efficiency. Modifications included the use of a proton sponge to protect acid-sensitive products [83] or adjustments such as lowering the temperature (from 0 °C to −15 °C) [84]. Further advancements included an enhanced Eckstein method, which employed bulkier 5′-phosphitylation reagents for more specific reactions, yielding 5′-NTPs in a one-pot, protection-free process [85]. Supported solution synthesis methods also emerged, focusing on reducing purification steps [2,86]. These approaches involve immobilizing substrates or phosphorylation agents onto insoluble resins or soluble polymers, making it easier to remove byproducts. However, attaching the base or sugar moiety of the nucleoside/nucleotide to the support limits this method to substrates without modifications to these parts. Additionally, further column purification is required to achieve product purity greater than 95%.

Several comprehensive reviews have explored the various chemical methods used in 5′-NTP synthesis over the decades and are recommended for further reading. Early works detail the first decades of research [87], while later reviews summarize chemical production processes and their applications [50,86,88]. More recent developments in the field are thoroughly discussed in newer publications [2,17].

Ongoing advancements in chemical methods have made it possible to efficiently and selectively produce a wide range of 5′-NTPs. However, some challenges persist, including the need for anhydrous reagents, multiple reaction steps, highly variable yields depending on substrate modifications, and the use of hazardous reagents and harsh conditions. No universal chemical method for 5′-NTP synthesis has been established yet, and for each desired product, the most suitable reaction route and conditions must be carefully evaluated.

2. Enzymatic Cascade Reactions to Produce Natural and Modified 5′-NTPs

To overcome the challenges of chemical synthesis routes, biochemists have worked on identifying and characterizing interesting enzyme candidates for biocatalytic 5′-NTP synthesis, as such an approach relies on the diversity and promiscuity of the applied enzymes. In publications on 5′-NTP synthesis processes, the main prejudice against biocatalytic routes was the limited substrate scope of natural enzymes, which could explain the reluctance of many chemists to consider them [89]. However, this widespread myth has been successfully disproved several times. A large number of cascade reactions have been developed that allow the efficient synthesis of a wide range of modified 5′-NTPs. One-pot cascade approaches starting from either nucleobases or nucleosides and using purified enzymes are described in detail in the following chapters, and the advantages and disadvantages are outlined. Two- or multistep procedures, or protocols combining chemical and enzymatic methods or cascades using enzyme extracts, are, however, not addressed in this review.

2.1. Cascades Starting from Nucleosides and Using Nucleoside and NMP Kinases

Our group has recently explored the biocatalytic production of a variety of natural and modified 5′-NTPs using pyrimidine or purine nucleosides as substrates in one-pot enzyme cascades involving NKs, NMPKs, and NDPKs [90] (Figure 3). By utilizing a minimal set of enzymes, we successfully synthesized 5′-NTPs with conversion rates ranging from 27% to over 99% (Figure 3) within 19 h in a modular approach (Figure 3C). Another research group applied a NK-NMPK cascade for ATP production from adenosine, achieving 94% conversion [91] (Figure 3B). However, this process required several days, with the addition of AMP and ADP as intermediates, and some degradation of the product was observed. Furthermore, 5F-CTP was synthesized from 5F-cytidine through a comparable kinase cascade, resulting in a 78% yield after 72 h [92] (Figure 3B).

While the methods above all utilized NKs and NMPKs for the first and the second phosphorylation step, the third step varied (Figure 3A). AcK was used in ATP synthesis [91], PK in 5F-CTP synthesis [92], and NDPK in our modular synthesis approach [90]. This highlights the flexibility in enzyme choice for the third reaction step. Moreover, AcK or PK not only catalyze 5′-NTP synthesis but also enables the regeneration of ATP, which was shown to have a positive impact on final product yields.

The 5′-NTP synthesis pathway utilizing NKs and NMPKs offers several advantages over alternative methods. It supports the production of a broad range of products, including N- and C-glycosidic nucleotides, as well as sugar- and base-modified variants (Figure 3). This approach requires only three enzymes and a cost-effective and easy-to-access nucleoside substrate, making it an efficient and versatile option for 5′-NTP synthesis. When a phosphate donor regeneration system is incorporated, high conversions are achieved [90,91,92].

2.2. Biocatalytic Synthesis of 5′-NTPs Starting from Nucleobases and Using NMP Kinases

In addition to nucleosides, nucleobases are an affordable starting material. PRTs are commonly used as catalysts to couple the nucleobase with the activated sugar moiety PRPP. However, this method cannot be used to synthesize cytosine derivatives, as no PRT is available for this purpose. Additionally, the synthesis of C-nucleotides requires the use of different enzymes, specifically PUS. The following sections describe cascades involving PRT/NMPK and PUS/NMPK.

By combining a PRT with adenylate kinase and CK, 2F-ATP was synthesized in 96 h with a 90% yield, starting with a nucleobase and ribose as substrates [66] (Figure 4). Ribose was first phosphorylated by ribokinase to form ribose-5-phosphate, which was then converted to PRPP by PRPP synthetase (Figure 4A). Similar cascades have also been used for the production of 5F-UTP [92] and 8-aza-GTP [64], yielding 80% and 60% product, respectively (Figure 4). In both studies, PK was employed to interconvert the 5′-NDP to the corresponding 5′-NTP. To ensure efficient conversion from PRPP to 5′-NMP, substrate-specific PRTs were utilized in each case. Adenine PRT was used for 2F-ATP synthesis [66], while uracil PRT and guanine PRT were applied to produce 5F-UTP [92] and 8-aza-GTP [64], respectively (Figure 4A).

The advantages of this approach include the potential for very high (>80%) conversions, particularly when phosphate donor regeneration systems are employed. Additionally, ribose and nucleobases are inexpensive starting materials. With the identification of 4-(D-ribofuranosyl)aminobenzene synthases (ß-RFAS), C-nucleosides could also be produced using this approach. ß-RFAS catalyze the formation of a β-C-ribosidic bond between PRPP and differing acceptor substrates. Depending on the enzyme, substrates may contain a carboxylic group. Examples are aromatic acceptors (e.g., para-aminobenzoic acid) or pyrazole-derived carboxylic acids. Detailed information on this enzyme class is available from Pfeiffer and Nidetzky (2023) [93]. However, the drawbacks include a comparably long route requiring five enzymes and the fact that these protocols are currently limited to ribose as the sugar moiety. Moreover, PRPP synthases are often inhibited by purine 5′-NDPs.

Another approach to access C-nucleotides is based on the preplacement of PRT with PUS (Figure 5). Several sugar- and base-modified pseudouridine-5′-monophosphate variants were produced and then phosphorylated to NTPs applying Escherichia coli pseudouridylate synthase YeiN, CMPK, and NDPK in a one-pot, multi-step process with yields of up to 85% [94] (Figure 5B). A PK-based ATP regeneration system was incorporated into the reaction. The process started from ribose, which was initially converted to ribose-5-phosphate, the substrate for PUS, by ribokinase (Figure 5A). Unlike other approaches, this method is specific to C-nucleotides and has so far only been applied to uracil derivatives [94].

2.3. Biocatalytic Synthesis of 5′-NTPs Using Polyphosphate Kinases

Due to the cost-effective phosphate donor, polyphosphate, polyphosphate kinases (PPKs) have gained significant importance in recent years. This is evident not only in ATP regeneration but also in 5′-NTP synthesis. PPK-based cascades, like NMPK-based cascades, begin with the nucleobase or nucleoside. To date, either two different PPKs have been used to convert 5′-NMPs to 5′-NDPs or 5′-NDPs to 5′-NTPs or a single PPK has been utilized to catalyze both reaction steps.

When synthesis starts from nucleobases, PRPP is used as an activated sugar, and a PRT serves as the biocatalyst (Figure 6A). Afterwards, the 5′-riboNTP was synthesized using Meiothermus ruber PPK or an optimized variant, and subsequently, the 5′-dNTP was formed through an RNR [80,95]. Using this approach, compounds such as cladribine-5′-TP and 6-mercapto-5′-dGTP have been formed, with conversions of 80% and 12%, respectively (Figure 6B). A 5′-NMP screening was also conducted for the PPKs used, confirming that both the Meiothermus ruber wildtype enzyme and the engineered version showed a broad substrate spectrum, converting compounds such as (d)AMP, (d)GMP, (d)CMP, 8-bromoAMP, and etheno-AMP into the respective 5′-NTPs [80]. A PRT-PPK-based cascade, without RNR, was also employed for the synthesis of 5-aminoimidazole-4-carboxamide 5′-NTP (5′-NTP of AICA) (Figure 6). Using the Meiothermus ruber PPK, a 65% yield was achieved in a two-enzyme approach [96].

Starting with nucleosides, phosphorylation by an NK is first performed, followed by the PPK-mediated conversion of 5′-NMP to 5′-NTP (Figure 7A). Sun et al. successfully synthesized ATP using this approach (Figure 7B) [97], while Bencic et al. investigated a range of compounds, including all natural 5′-NTPs and base-modified variants, achieving yields from 8% to 72% (Figure 7B) [79].

NK-PPK cascades have also been used in larger cascades. For example, ATP and GTP building blocks were efficiently produced for the subsequent synthesis of 2′,3′-cGAMP [98,99]. Furthermore, the route was employed to establish a SAM regeneration system, where ATP was efficiently generated from adenosine in a cascade involving Saccharomyces cerevisiae adenosine kinase, Acinetobacter johnsonii PPK, and Sinorhizobium meliloti PPK [100,101].

In summary, these studies demonstrate the great potential of PPK-containing enzyme cascades, as PPKs possess a broad substrate spectrum. However, it remains to be seen how well this system can be applied to sugar-modified compounds or whether the PPK-typical hyperphosphorylation of nucleotides will present a challenge in downstream processing.

2.4. Other Interesting Approaches to Produce 5′-NTPs

An innovative approach to synthesizing 5′-NTPs involved the development of a large enzyme cascade inspired by the de novo purine biosynthesis pathway. Using simple building blocks like glucose, CO₂, NH₃, and serine, natural purine nucleotides ATP and GTP were synthesized with up to 66% isolated yield [102]. This complex process includes CK in the final step and allows for specific isotopic labeling at various positions of the 5′-NTP products. Similarly, the same group developed an 18-enzyme cascade to produce isotopically labeled pyrimidines CTP and UTP from glucose, hydrogen carbonate, ammonia, and aspartate [103]. Although this method relies on simple starting materials, the use of over 15 enzymes makes it practical mainly for introducing specific isotopic labels.

A different approach for 5′-dNTP synthesis involves a mutated nucleoside 2′-deoxyribosyltransferase (NdT), engineered to transfer nucleobases not only between deoxynucleosides but also between 5´-dNMPs, 5´-dNDPs and 5´-dNTPs [104]. This enzyme was used to produce base-modified purine and pyrimidine 5′-dNTPs from their respective nucleobases and CTP (Figure 8). However, this method is limited to N-glycosidic 5´-dNTPs and has so far resulted in low conversion rates.

3. Importance of ATP-Regeneration Systems in the Synthesis of 5′-NTPs

A key parameter for cofactor-dependent enzymes is the total turnover number (TTN), which indicates the moles of product generated per mole of cofactor over its entire lifespan, serving as a measure of catalytic efficiency. In the absence of a regeneration system for group-transfer-cofactors like ATP, these cofactors must be used in stoichiometric amounts, making the process inefficient for industrial applications. However, by implementing a regeneration system, the TTN can be increased to values of ≥10⁵ [5]. A recent analysis highlighted the efficiency of ATP regeneration systems across various reactions [105]. Various ATP regeneration systems, such as PK/phosphoenolpyruvate, AcK/acetyl phosphate, and PPK/polyphosphate, are frequently employed. While PK/phosphoenolpyruvate and AcK/acetyl phosphate have been used for decades, the PPK/polyphosphate system is gaining increasing attention for its cost-effectiveness only recently [59,79,92,97]. Although Escherichia coli PPK was first mentioned for ATP regeneration in the 1950s [74], it was not until several decades later that interest in its role in metabolism and nucleotide synthesis grew significantly.

Each ATP regeneration system has distinct advantages and drawbacks depending on the enzyme and phosphate donor used. Acetyl phosphate is an affordable and widely used phosphate donor. It can be easily synthesized from inexpensive materials and used as a crude solution in some applications [42]. However, it is less stable than alternatives [106]. To address this issue, a pyruvate oxidase-based synthesis of acetyl phosphate was developed. To overcome the limitations of this method, including hydrogen peroxide formation and poor oxygen mass transfer from the gas to the liquid phase, an electrochemical oxidation of FADH₂, the cofactor of pyruvate oxidase, has recently been proposed [107]. Another drawback of AcKs is their sensitivity to oxidation, which necessitates chemical protection to prevent inactivation [106].

In contrast, phosphoenolpyruvate is highly stable and thermodynamically the most efficient phosphate donor due to its high free enthalpy of phosphate hydrolysis, which results in nearly irreversible phosphate transfer to an acceptor [5,106]. However, its industrial use is limited by its relatively high cost and a potential kinase inhibition by pyruvate at concentrations around 10 mM [106].

Polyphosphate has become increasingly popular as a secondary phosphate donor due to its low cost and stability across a broad pH range, though it has some limitations [70]. Many commercial polyphosphate products consist of mixtures with varying chain lengths [108], which can complicate studies that require a specific chain length, particularly if the enzyme used has a preference. Additionally, polyphosphate and free phosphate can precipitate with magnesium or other essential cations, potentially affecting enzyme activity. Furthermore, polyphosphates′ lower free enthalpy values compared to ATP result in lower equilibrium constants, requiring a large excess of polyphosphate for efficient reactions [70].

Another ATP regeneration system, using CK and phosphocreatine, has also been explored. Phosphocreatine, like acetyl phosphate, is inexpensive but unstable [106]. CK has a low K_m for ADP, which is advantageous for certain applications [70].

Phosphate donor regeneration systems are incorporated into enzymatic reactions for several reasons. In some cases, they prevent enzyme inhibition caused by the accumulation of side products like ADP [109] or UDP [110]. Hughes et al. also observed a slight acceleration of the reaction when PK was added. Economically, it is often beneficial to use substoichiometric amounts of phosphate donor, which are continuously regenerated from a cheaper secondary source, ensuring efficient substrate phosphorylation [92,111,112]. ATP minimization is particularly important in 5′-NTP synthesis, as the structural similarity between the phosphate donor and the target product can complicate chromatographic separation. Although Davisson’s group reported improved product yields with this approach [59], observations were not quantified. In our study on the impact of the PK/phosphoenolpyruvate system on 5′-NTP synthesis [90] we found that continuous regeneration of phosphate donor (d)ATP from (d)ADP was essential for improved 5′-NTP production. The implementation of the PK/phosphoenolpyruvate system led to a significant increase in product conversion, with a four- to nine-fold improvement for natural nucleotides and a four- to six-fold increase for sugar- or base-modified nucleosides. Additionally, reaction kinetics were also enhanced.

4. Conclusions

The growing demand for natural 5′-NTPs and modified analogs calls for a simpler, more cost-effective, and sustainable production method compared to current multi-step chemical syntheses. Over the past few decades, promising biocatalytic cascade processes have been developed to produce a diverse range of natural and modified 5′-NTPs, using affordable nucleosides or nucleobases as substrates. The field has experienced a renaissance due to the coronavirus pandemic and the increasing demand for 5′-NTPs in diagnostics and for mRNA vaccines. Several efficient methods for synthesizing 5′-NTPs are now available, demonstrating that a major skepticism towards enzymatic processes in this area is no longer valid: natural enzymes can be used to produce a wide variety of 5′-NTP analogs. The next step is now to demonstrate that these approaches can be scaled up to meet industrial needs. However, scaling up is promising, as the synthesis of pseudouridine 5′-monophosphate has already been successfully demonstrated at a 600 mM scale [26]. Enzymatic processes are expected to play a key role in the pharmaceutical and chemical industries, especially in driving more sustainable solutions.